Basophil Expression Research Articles

Advances in the pathogenesis representing definite outcomes in chronic urticaria.

We reviewed in this article, the recent advances in CSU physiopathology and potential clinical and laboratory biomarkers in CSU. In addition to the central role of mast cells in urticaria physiopathology, increased interest in basophils has arisen. Recent data corroborate the autoimmunity pathway as one of the main pathways in mast cell activation. The association of inflammatory cytokines, heat shock proteins and staphylococcal infection with CSU are also reviewed. C-reactive protein, D-dimers, autologous serum skin test, IgE levels and FcεRI expression in basophils have shown their potential as biomarkers for disease duration, activity, severity and/or response to treatment. A comprehensive understanding of chronic spontaneous urticaria mechanisms is essential to find novel biomarkers and treatments. The use of these biomarkers in clinical practice will guide us in choosing the best treatment option for our patients.

Eosinophils Mediate Basophil-Dependent Allergic Skin Inflammation in Mice

The mechanisms leading to allergic skin inflammation, such as atopic dermatitis or urticaria, are poorly defined. Here we used a mouse model for IgE-dependent chronic allergic inflammation to study the role of basophils and eosinophils for induction of pathology. FcεRI expression in basophils was required for the ear swelling response, and basophils promoted the expression of eosinophil-recruiting chemokines in the ear. The ear swelling response could be prevented by prior infection of mice with helminths in an IgE-dependent manner. Impaired skin eosinophilia and reduced ear swelling was further observed in IL-4/IL-13-deficient and STAT6-deficient mice. In addition, eosinophil-deficient ΔdblGATA mice showed only weak ear swelling response, which could be enhanced by eosinophil transfer. This suggests that IgE-activated basophils orchestrate the recruitment of eosinophils by secretion of IL-4/IL-13, which leads to STAT6-dependent expression of CCL24 from endothelial cells and extravasation of eosinophils into the ear pinna. Eosinophils are then the critical effector cells that cause pathology. Therefore, combined therapeutic approaches that block basophil activation and reduce eosinophil numbers could be efficient strategies to improve treatment of chronic allergic disorders of the skin.

Canis familiaris allergen Can f 7: Expression, purification and analysis of B cell epitopes in Chinese children with dog allergies.

Dogs are a major source of indoor allergens. However, the prevalence of dog allergies in China remains unclear, especially in children. In the present study, Can f 7, a canine allergen belonging to the Niemann pick type C2 protein family, was selected to study its sensitization rate in Chinese children with dog allergies. The Can f 7 gene was subcloned into a pET‑28a vector and expressed in Escherichia coli BL21 (DE3) cells. Recombinant Can f 7 was purified by nickel affinity chromatography, identified by SDS‑PAGE electrophoresis, and had its allergenicity assessed by western blot, ELISA and basophil activation tests. Through a series of bioinformatical approaches, B‑cell epitopes, secondary structures, and 3 dimensional (3D) homology modeling of Can f 7 were predicted. The activity of the B cell epitopes was verified by ELISA. The recombinant Can f 7 showed a distinct band with a molecular weight of 14 kDa. Six of 20 sera from dog‑allergic children reacted positively to the Can f 7. Can f 7 induced an ~4.0‑fold increase in cluster of differentiation 63 and C‑C motif chemokine receptor R3 expression in basophils sensitized with the serum of dog‑allergic children compared with those of non‑allergic controls. The secondary structure analysis showed that Can f 7 contains 6 β‑sheets. Five B cell epitopes of Can f 7 were predicted, and two of these were confirmed by ELISA. These results indicate that Can f 7 is an important canine allergen in Chinese children and provide novel data for further research concerning the use of Can f 7 in the diagnosis and treatment of Chinese children with canine allergy symptoms.

Evaluation of the basophil activation test and skin prick testing for the diagnosis of sesame food allergy.

The prevalence of sesame food allergy (SFA) has increased over recent years, with the potential of anaphylactic reactions upon exposure. Oral food challenge (OFC) remains the diagnostic standard, yet its implementation may be risky. Commercial skin prick tests (SPT) have a low sensitivity. Investigation of alternate diagnostic methods is warranted. To evaluate the utility of SPT and the basophil activation test (BAT) for SFA diagnosis. Eighty-two patients with suspected SFA completed an open OFC to sesame or reported a recent confirmed reaction. Patients were administered skin prick tests (SPT) with commercial sesame seed extract (CSSE) and a high protein concentration sesame extract (HPSE) (100 mg/mL protein). Whole blood from 80 patients was stimulated with sesame seed extract (40-10 000 ng/mL protein) for BAT), assessing CD63 and CD203c as activation markers. Sixty patients (73%) had IgE-mediated reactions to sesame, and 22 (27%) did not react. Receiver operating characteristic (ROC) curve analysis demonstrated an area under the curve (AUC) of 0.87 for HPSE-SPT and 0.66 for CSSE-SPT. At 1000 ng/mL of sesame protein, induction of CD63 and CD203c was weakly but significantly associated with OFC eliciting dose by rank (Spearman's rho = -.42 (P < .01) and -.35 (P < .05) for CD63 and CD203c, respectively). By ROC analysis, the AUC was 0.86 for CD63 and was 0.81 for CD203c sesame-induced basophil expression. Using HPSE-SPT as a first test to definitively diagnose (n = 24) or rule-out (n = 5) SFA and BAT as a second test to diagnose the remainder results in the correct classification of 73 of 80 (91%) patients, leaving one false negative and 4 false positive patients. Two BAT non-responders remain unclassified by this algorithm. While prospective cohort validation is necessary, joint utilization of BAT and SPT with HPSE extract may obviate the need for OFC in most SFA patients.

Autoantibodies to IgE and FcεRI and the natural variability of spleen tyrosine kinase expression in basophils

Secretion from human basophils and mast cells requires spleen tyrosine kinase (SYK) activity, but SYK expression is highly variable in the general population, and this variability predicts the magnitude of IgE-mediated secretion. One known mechanism of modulating SYK expression in human basophils is aggregation of FcεRI. This study examines the possibility that functional autoantibodies are present in a wide variety of subjects and, in particular, subjects whose basophils poorly express SYK. It also tests whether any found antibodies could modulate SYK expression in maturing basophils and whether interaction with FcγRIIb/CD32b modulates the effect. An experimental algorithm for classifying the nature of histamine release induced by serum from 3 classes of subjects was developed. The frequency of functional autoantibodies that produce characteristics concordant with FcεRI-mediated secretion was zero in 34 subjects without chronic spontaneous urticaria (CSU). In patients with CSU, the frequency was lower than expected, approximately 7%. For the 5 of 68 unique sera from patients with CSU tested that contained anti-FcεRI or anti-IgE antibodies, these antibodies were found to induce downregulation of SYK in both peripheral blood basophils and basophils developed from CD34+ progenitors. Blocking interaction of these antibodies with CD32b did not alter their ability to downregulate SYK expression. This study establishes that functional autoantibodies to IgE/FcεRI do not provide a good explanation for the variability in SYK expression in basophils in the general population. They do show that if antibodies with these characteristics are present, they are capable of modulating SYK expression in developing basophils.

IL-3 but not monomeric IgE regulates Fc\u03b5RI levels and cell survival in primary human basophils

Binding of allergen-specific IgE to its primary receptor FcεRI on basophils and mast cells represents a central event in the development of allergic diseases. The high-affinity interaction between IgE and FcεRI results in permanent sensitization of these allergic effector cells and critically regulates their release of pro-inflammatory mediators upon IgE cross-linking by allergens. In addition, binding of monomeric IgE has been reported to actively regulate FcεRI surface levels and promote survival of mast cells in the absence of allergen through the induction of autocrine cytokine secretion including interleukin-3 (IL-3). As basophils and mast cells share many biological commonalities we sought to assess the role of monomeric IgE binding and IL-3 signaling in FcεRI regulation and cell survival of primary human basophils. FcεRI cell surface levels and survival of isolated blood basophils were assessed upon addition of monomeric IgE or physiologic removal of endogenous cell-bound IgE with a disruptive IgE inhibitor by flow cytometry. We further determined basophil cell numbers in both low and high serum IgE blood donors and mice that are either sufficient or deficient for FcεRI. Ultimately, we investigated the effect of IL-3 on basophil surface FcεRI levels by protein and gene expression analysis. Surface levels of FcεRI were passively stabilized but not actively upregulated in the presence of monomeric IgE. In contrast to previous observations with mast cells, monomeric IgE binding did not enhance basophil survival. Interestingly, we found that IL-3 transcriptionally regulates surface levels of FcεRI in human primary basophils. Our data suggest that IL-3 but not monomeric IgE regulates FcεRI expression and cell survival in primary human basophils. Thus, blocking of IL-3 signaling in allergic effector cells might represent an interesting approach to diminish surface FcεRI levels and to prevent prolonged cell survival in allergic inflammation.

Basophils activated via TLR signaling may contribute to pathophysiology of type 1 autoimmune pancreatitis

BackgroundPathophysiology of type 1 autoimmune pancreatitis (AIP) is still unclear. We previously reported that M2 macrophages might play an important role in type 1 AIP. Recently, it has been reported that basophils regulate differentiation to M2 macrophages. In this study, we investigated basophils from the pancreatic tissue and peripheral blood of individuals with type 1 AIP.MethodsBy using immunohistochemistry, we investigated basophils in pancreatic tissue from 13 patients with type 1 AIP and examined expression of toll-like receptors (TLRs) by these cells. Additionally, we obtained peripheral blood samples from 27 healthy subjects, 40 patients with type 1 AIP, 8 patients with alcoholic chronic pancreatitis, 10 patients with bronchial asthma, and 10 patients with atopic dermatitis, and analyzed activation of basophils by stimulating them with ligands of TLR1–9. We also compared TLR expression in basophils from the tissue and blood samples.ResultsBasophils were detected in pancreatic tissues from 10 of 13 patients with type 1 AIP. Flow cytometric analysis revealed that the ratios of basophils activated by TLR4 stimulation in type 1 AIP (9.875 ± 1.148%) and atopic dermatitis (11.768 ± 1.899%) were significantly higher than those in healthy subjects (5.051 ± 0.730%; P < 0.05). Levels of basophils activated by TLR2 stimulation were higher in seven type 1 AIP cases. Furthermore, stimulation of TLR2 and/or TLR4, which were expressed by basophils in pancreas, activated basophils in peripheral blood.ConclusionsBasophils activated via TLR signaling may play an important role in the pathophysiology of type 1 AIP.

Leukocyte and plasma activation profiles in chronically transfused patients with a history of allergic reactions.

Allergic transfusion reactions are drawbacks to the benefits of transfusion. Classically, allergic transfusion reactions depend on histamine release from mast cells or basophils, but other leukocyte subsets may also be important. Thus, we propose to better define the exact leukocyte subsets involved in allergic transfusion reactions. The overall objective of the current study was to compare the activation of specific peripheral blood leukocyte subsets (monocytes, neutrophils, eosinophils, and basophils) in a cohort of 13 patients who received chronic transfusions and had a history of allergic transfusion reactions compared with a control group of patients who received chronic transfusions and had no history of allergic transfusion reactions. Leukocyte subsets were analyzed by flow cytometry at baseline and after red blood cell transfusion, and cytokine levels in platelet-free plasma collected at the same time points were measured by Luminex assay. Flow cytometry and cytokine profiles before and after transfusion did not differ significantly between patients who did and did not have a history of allergic transfusion reactions (p > 0.05). However, post-transfusion samples from both groups showed a decrease in CD63 expression in basophils, monocytes, and eosinophils and a decrease in CD45 expression in all leukocyte subsets compared with pretransfusion samples. Interleukin 10 levels increased after transfusion in the group with a history of allergic transfusion reactions (p = 0.0469), and RANTES (regulated upon activation, normal T-cell expressed and secreted) was significantly decreased post-transfusion in all patients (p = 0.0122). None of the leukocyte subsets from patients who had a history of allergic transfusion reactions significantly increased in activation either before or after transfusion. All leukocyte subsets from patients who did and did not have a history of allergic transfusion reactions decreased in their activation profile upon transfusion challenge.

Syk expression and IgE-mediated histamine release in basophils as biomarkers for predicting the clinical efficacy of omalizumab

Syk expression and IgE-mediated histamine release in basophils as biomarkers for predicting the clinical efficacy of omalizumab

Use of Biomarker Based on Human Basophil Syk Expression to Predict the Clinical Efficacy of Omalizumab Treatment in Allergic Asthmatics

Treatment with omalizumab results in the increased sensitivity of basophils to IgE-mediated stimulation, likely due to an increase in syk expression in basophils. Studies in patients with peanut allergy suggested that the increased sensitivity works in opposition to the therapeutic benefits of reduced free IgE. This study examined whether a priori knowledge of syk expression or maximum IgE-mediated histamine release, which is related to syk expression, would provide a predictor of the efficacy of omalizumab in patients with allergic asthma. Adult asthma patients were enrolled and treated with omalizumab for 16 weeks using FDA-approved dosing criteria. Blood basophils were examined for syk expression, IgE-mediated histamine release and changes in these two parameters before and after treatment. Clinical response was measured via a physician’s assessment score (PAS, 1-5 score range, 1 = minimal symptoms) and a final assessment of success. Basophil studies and clinical evaluations were maintained by separate groups. Ten patients satisfied entry criteria (PAS scores of ≥4). For this group, all four metrics, starting syk, changes in syk, starting and changes in histamine release showed a positive chi-square association (p-values ranged 0.0098 to 0.038) with the success of treatment. This study suggests that changes in syk expression and therefore the functionality of human basophils may provide a predictor of successful treatment of asthmatics with omalizumab. Since the change in syk expression is related to starting syk, it is possible that baseline basophil syk or histamine release might be a bio-marker of successful treatment with omalizumab.

Exploratory analysis of CD63 and CD203c expression in basophils from hazelnut sensitized and allergic individuals.

BackgroundSensitization to hazelnut (HN) is frequent and requires clarification to determine whether this sensitization is clinically relevant. The aim of this study was to investigate basophil activation profiles in HN-sensitized and allergic subjects.MethodsBasophil activation was determined by flow cytometric analyses of CD63 and CD203c expression using several HN allergen concentrations. Depending on their clinical reaction pattern, an oral allergy symptom group (OAS, n = 20), a systemic reaction group (n = 12) and a sensitized group without clinical symptoms (n = 20) were identified. Additionally, 10 non-allergic and non-sensitized individuals served as controls.ResultsCD63 and CD203c expression differed between allergic (OAS and systemic group) and sensitized subjects. The HN concentration required to activate 30% of CD203c+ basophils [effective concentration (EC)30] was significantly higher in sensitized versus the allergic group (p = 0.0089). This was more pronounced when the basophil allergen threshold sensitivity (CD-sens) was calculated (CD63: p = 0.018; CD203c: p = 0.009).ConclusionOur data indicate that the basophil activation test may provide information to better distinguish between sensitized and allergic subjects if several allergen concentrations are considered. CD203c expression displayed a better discrimination compared to CD63; therefore, its diagnostic value might be superior compared with CD63.Electronic supplementary materialThe online version of this article (doi:10.1186/s13601-016-0134-7) contains supplementary material, which is available to authorized users.

A randomized multicenter study evaluating Xolair persistence of response after long-term therapy

Few data are available to assist clinicians with decisions regarding long-term use of asthma therapies, including omalizumab. We sought to evaluate the benefit and persistence of response in subjects continuing or withdrawing from long-term omalizumab treatment. Evaluating the Xolair Persistency Of Response After Long-Term Therapy (XPORT) was a randomized, double-blind, placebo-controlled withdrawal study that included subjects with moderate-to-severe persistent asthma receiving long-term omalizumab. Subjects were randomized by using a hierarchical dynamic randomization scheme to continue their same dose of omalizumab or withdraw to placebo and were then followed every 4weeks for 1year. The primary outcome was any protocol-defined severe asthma exacerbation. The secondary outcome was time to first protocol-defined severe asthma exacerbation. Exploratory outcomes included changes in Asthma Control Questionnaire and Asthma Control Test scores. Significantly more subjects in the omalizumab group (67%) had no protocol-defined exacerbation than in the placebo group (47.7%); an absolute difference of 19.3% (95% CI, 5.0%, 33.6%) represents a 40.1% relative difference. Time to first protocol-defined exacerbation analysis revealed a significantly different between-group exacerbation pattern that was consistent with the primary analysis. Subjects continuing omalizumab had significantly better asthma control (mean [SD] change from baseline to week 52: Asthma Control Test score, -1.16 [4.14] vs placebo, -2.88 [5.38], P=.0188; Asthma Control Questionnaire score, 0.22 [0.66] vs placebo, 0.63 [1.13], P=.0039). Discontinuation of omalizumab was associated with an increase in free IgE levels and an increase in basophil expression of the high-affinity IgE receptor. No safety concerns were noted. Continuation of omalizumab after long-term treatment results in continued benefit, as evidenced by improved symptom control and reduced exacerbation risk.

Basophil CD63 expression in chronic spontaneous urticaria: correlation with allergic sensitization, serum autoreactivity and basophil reactivity.

The underlying causes and factors contributing to the disease severity of chronic spontaneous urticaria (CSU) are unknown. Given the important role of basophils in the pathogenesis of urticaria and that CD63 serves as a useful marker for basophil activation and detecting, CD63 expression of basophils is a reliable tool for diagnosing allergy and hypersensitivity reactions to different allergens; the objective of this study was to investigate whether the level of basophil CD63 expression is correlated with allergen sensitization, serum autoreactivity and basophil reactivity in patients with CSU. Basophil-enriched leucocytes were separated from the blood of 64 patients with chronic urticaria (54 CSU patients and 10 symptomatic dermographism patients), 18 healthy control subjects and seven atopic donors without urticaria. Flow cytometry was then used to detect CD63 expression on the cell membrane of basophils from all samples. Analysis was also preformed on basophils incubated with sera from CSU patients with positive or negative autologous serum skin test (ASST). CD63 expression was significantly higher in the basophils from patients with CSU than in those from patients with symptomatic dermographism and the healthy control group. The levels of CD63 expression in CSU patients with ASST+ and/or allergen sensitization were higher than those with ASST- and/or no allergen sensitization patients. Incubation with ASST+ serum resulted in an increased expression of CD63 in the basophils of ASST+ CSU patients, whereas no such response was observed in healthy controls or ASST- CSU patients. The increased CD63 expression in basophils from CSU patients may correlate with allergen sensitization, autoreactivity of serum and basophil reactivity. Our results suggest that CD63 may contribute new insight into the pathogenesis of CSU.

Stability of Syk protein and mRNA in human peripheral blood basophils.

In human basophils, Syk expression is 10-fold lower than most other types of leukocytes. There are indirect studies that suggest that Syk protein is highly unstable (a calculated half-life less than 15 min) in human peripheral blood basophils. Therefore, in these studies, Syk stability was directly examined. Purified basophils were metabolically labeled and a pulse-chase experimental design showed Syk protein to be stable in the time frame of 12 h (95% likelihood that half-life is more than 12 h). However, its synthetic rate was very slow (∼10-fold slower) compared with CD34-derived basophils, which have been shown to express levels of Syk consistent with other mature circulating leukocytes. Syk mRNA expression was found to be 5-30-fold lower than other cell types (CD34-derived basophils, peripheral blood eosinophils, and plasmacytoid dendritic cells). Syk protein and mRNA levels, across cell types, were relatively concordant. Syk mRNA in basophils showed a half-life of 3.5 h, which was greater than that of interleukin-4 or Fc epsilon receptor I-α mRNA (∼2 h), but somewhat shorter than Fc epsilon receptor I-β mRNA (8 h). A comparison of miR expression between CD34-derived and peripheral blood basophils demonstrated only 1 significant increase, in miR-150 (77-fold). Transfection in human embryonic kidney cells of a stabilized form of miR-150 showed that it modified expression of c-Myb mRNA but not of Syk mRNA or protein. These results suggest that low Syk expression in basophils results, not from protein instability and perhaps not from mRNA stability. Instead, the results point to the transcriptional nature of an important point of regulation.

Serum from a subset of patients with chronic lymphocytic leukaemia and large local reactions to mosquito bites significantly induces upregulation of CD63 surface expression in atopic basophils

Serum from a subset of patients with chronic lymphocytic leukaemia and large local reactions to mosquito bites significantly induces upregulation of CD63 surface expression in atopic basophils

IL-25 and IL-33 induce Type 2 inflammation in basophils from subjects with allergic asthma.

BackgroundThe alarmin cytokines IL-25 and IL-33 are key promoters of type 2 inflammation. Basophils respond to alarmin cytokines, however the relationship of these cytokines with basophil activation and recruitment in human studies of allergic asthma has not been well characterized. This study investigated the effect of IL-25 and IL-33 on basophils in a model of allergic asthma.Methods10 mild allergic asthmatics underwent allergen and diluent inhalation challenges. Bone marrow aspirates were collected at pre-challenge and 24 h (h) post challenge. Peripheral blood and sputum samples were collected at pre-challenge, 7 h, and 24 h post-challenge to measure basophil expression of IL-17RB, ST2, and intracellular IL-25. Freshly isolated peripheral blood basophils from allergic donors were incubated overnight with IL-25 and IL-33, or sputum supernatant collected post-allergen to assess pro-inflammatory effects of mediators released in the airways.ResultsThere were increased percentage of basophils expressing IL-17RB, ST2, and intracellular IL-25 collected from bone marrow, peripheral blood, and sputum after allergen inhalation challenge. In vitro stimulation with IL-25 and IL-33 increased the percentage of basophils expressing intracellular type 2 cytokines and surface activation markers, and primed eotaxin-induced migratory potential of basophils, which was mediated directly through IL-17RB and ST2, respectively. Stimulation of basophils with sputum supernatants collected post-allergen challenge up-regulated the percentage of basophils expressing markers of activation and intracellular type 2 cytokines, which was reversed following blockade of the common β chain (βc).ConclusionsOur findings indicate that the alarmin cytokines IL-33 and IL-25 increase basophil activation and migratory potential, and may pose as a novel therapeutic targets for the treatment of allergic asthma.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-016-0321-z) contains supplementary material, which is available to authorized users.

Highly increased levels of IgE antibodies to vaccine components in children with influenza vaccine–associated anaphylaxis

Influenza vaccines produced in embryonated eggs might pose a risk to patients with egg allergy. However, patients experiencing influenza vaccine-associated anaphylaxis (IVA) do not always have egg allergy. In the 2011-2012 season, an unusually high incidence of IVA was reported in Japan. We sought to identify the cause of the increase in anaphylactic events in 2011-2012 in Japan. We collected blood specimens from patients with IVA from all areas of Japan. We analyzed 19 patients with confirmed IVA and 25 age-matched control subjects, including 10 with egg allergy who had no adverse events after corresponding vaccination. ELISA was used to measure specific IgE levels to the trivalent vaccines of several manufacturers and hemagglutinin proteins derived from both egg and cell cultures. Antigen-induced basophil activation was evaluated by measuring CD203c expression by means of flow cytometry. Vaccine excipients were also examined for effects on CD203c expression. None of the patients with IVA had severe egg allergy. Levels of specific IgE antibodies to influenza vaccine antigens, whole-vaccine products from different manufacturers, and hemagglutinin proteins (A H1, H3, and B) derived from both egg and cell cultures were significantly increased in patients with IVA compared with those in control subjects. Influenza vaccine-induced CD203c expression in basophils was also highly enhanced in patients with IVA but not in control subjects. Because IVA was most frequent in patients who received 2-phenoxyethanol (2-PE)-containing vaccine, the effect of this preservative on basophil activation was examined, and the activation was slightly enhanced by 2-PE but not thimerosal. The 2011-2012 IVA spike in Japan was caused by specific IgE antibodies to influenza vaccine components. Excipients could not be implicated, except for a modest effect of 2-PE.

The STAT5-GATA2 pathway is critical in basophil and mast cell differentiation and maintenance.

Transcription factor GATA binding protein 2 (GATA2) plays critical roles in hematopoietic stem cell survival and proliferation, granulocyte-monocyte progenitor differentiation, and basophil and mast cell differentiation. However, precise roles of GATA2 in basophil and mast cell differentiation and maintenance have not been delineated. We have identified GATA2 as an essential transcription factor in differentiation of newly identified common basophil and mast cell progenitors into basophils and mast cells. We observed Gata2 haploinsufficiency for mast cell differentiation, but not for basophil differentiation. We examined the precise role of GATA2 in maintaining the expression of a wide range of genes that are important for performing basophil or mast cell functions. The effects of GATA2 on gene expression were broadly based. We demonstrated that GATA2 was required for maintaining Fcer1a mRNA and FcεRIα protein expression on both basophils and mast cells, as well as for maintaining Kit mRNA and c-Kit protein expression on mast cells. GATA2 was required for histamine synthesis and was also critical for Il4 mRNA expression in basophils and Il13 mRNA expression in mast cells. We demonstrate a STAT5-GATA2 connection, showing that the STAT5 transcription factor directly bound to the promoter and an intronic region of the Gata2 gene. Overexpression of the Gata2 gene was sufficient to direct basophil and mast cell differentiation in the absence of the Stat5 gene. Our study reveals that the STAT5-GATA2 pathway is critical for basophil and mast cell differentiation and maintenance.

Bacterial flagellin induces IL-6 expression in human basophils

Binding of allergen to IgE on basophils positively affects allergic inflammation by releasing inflammatory mediators. Recently, basophils were shown to express pattern-recognition receptors, such as toll-like receptors (TLRs), for recognizing microbe-associated molecular patterns (MAMPs) that are independent of allergen-IgE binding. In this study, we investigated whether MAMP alone can induce IL-6 production in a human basophil cell line, KU812. Stimulation with flagellin in the absence of allergen-IgE association induced IL-6 expression in KU812 cells, while stimulation with lipoteichoic acid, peptidoglycan, or poly I:C did not under the same condition. Flagellin-induced IL-6 expression was also observed in human primary basophils. Flow cytometric analysis showed that KU812 cells expressed flagellin-recognizing TLR5 both on the cell surface and in the cytoplasm while TLR2 and TLR3 were observed only in the cytoplasm. We further demonstrated that although flagellin augmented the phosphorylation of mitogen-activated protein kinases including p38 kinase, ERK, and JNK, flagellin-induced IL-6 production was attenuated by inhibitors for p38 kinase and ERK, but not by JNK inhibitors. In addition, flagellin enhanced phosphorylation of signaling molecules including CREB, PKCδ, and AKT. The inhibitors for PKA and PKC also showed inhibitory effects. Interestingly, flagellin-induced IL-6 production was further enhanced by pretreatment with inhibitors for PI3K, implying that PI3K negatively affects the flagellin-induced IL-6 production. Furthermore, DNA binding activities of NF-κB, AP-1, and CREB, which play pivotal roles in the induction of IL-6 gene expression, were increased by flagellin. These results suggest that flagellin alone is sufficient to induce IL-6 gene expression via TLR5 signaling pathways in human basophils.

Basophil expression of diamine oxidase: A novel biomarker of allergen immunotherapy response

Immunotherapy inhibits basophil histamine release, but the assay is cumbersome, and no one has studied the effects of immunotherapy withdrawal. Intracellular fluorochrome-labeled diamine oxidase (DAO) was used as a novel functional readout of basophil histamine release after immunotherapy. Results were compared with conventional basophil surface expression of activation markers. Subcutaneous immunotherapy (SCIT)-treated patients (n= 14), sublingual immunotherapy (SLIT)-treated patients (n= 12), participants who completed 3 years of treatment with grass pollen sublingual immunotherapy (the SLIT-TOL group; n= 6), patients with untreated seasonal allergic rhinitis (SAR; n= 24), and nonatopic control subjects (n= 12) were studied. Intracellularly labeled DAO(+) and surface expression of CD203c(bright), CD63(+), and CD107a(+) on chemoattractant receptor-homologous molecule expressed on TH2 lymphocytes (CRTh2)-positive basophils were measured by means of flow cytometry. Serum IgG4 levels and serum inhibitory activity for IgE-allergen complex binding to B cells (IgE-FAB) and basophil histamine release were also determined. Proportions of allergen-stimulated DAO(+)CRTh2(+) basophils were higher in participants in the SCIT, SLIT, and SLIT-TOL groups (all P< .0001) compared with those in patients in the SAR group. Similarly, there were lower proportions of CRTh2(+) basophils expressing surface CD203c(bright) (all P< .001), CD63 (all P< .001), and CD107a (allP< .01). Rhinitis symptoms were lower in the SCIT, SLIT, and SLIT-TOL groups (P< .001) compared with those in the SAR group. Serum inhibitory activity for IgE-FAB and basophil histamine release were also significantly greater in all immunotherapy groups (P< .05) compared with the SAR group. These results support long-term clinical and immunologic tolerance during and after grass pollen immunotherapy. Intracellularly labeled DAO expression by basophils merits further investigation as a surrogate biomarker for monitoring efficacy and tolerance after immunotherapy.